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WO1990011369A1 - Diagnostic en phase solide de conditions medicales - Google Patents

Diagnostic en phase solide de conditions medicales Download PDF

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Publication number
WO1990011369A1
WO1990011369A1 PCT/EP1990/000454 EP9000454W WO9011369A1 WO 1990011369 A1 WO1990011369 A1 WO 1990011369A1 EP 9000454 W EP9000454 W EP 9000454W WO 9011369 A1 WO9011369 A1 WO 9011369A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
primer
label
pcr
amplified
Prior art date
Application number
PCT/EP1990/000454
Other languages
English (en)
Inventor
Mathias Uhlen
Original Assignee
Cemu Bioteknik Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB898906642A external-priority patent/GB8906642D0/en
Priority claimed from GB898906641A external-priority patent/GB8906641D0/en
Priority to KR1019900702461A priority Critical patent/KR920700360A/ko
Application filed by Cemu Bioteknik Ab filed Critical Cemu Bioteknik Ab
Priority to EP90904803A priority patent/EP0464067B1/fr
Priority to DE69029726T priority patent/DE69029726T2/de
Publication of WO1990011369A1 publication Critical patent/WO1990011369A1/fr

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Classifications

    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
    • F16C—SHAFTS; FLEXIBLE SHAFTS; ELEMENTS OR CRANKSHAFT MECHANISMS; ROTARY BODIES OTHER THAN GEARING ELEMENTS; BEARINGS
    • F16C33/00—Parts of bearings; Special methods for making bearings or parts thereof
    • F16C33/02—Parts of sliding-contact bearings
    • F16C33/04—Brasses; Bushes; Linings
    • F16C33/20—Sliding surface consisting mainly of plastics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features

Definitions

  • This invention relates to a solid phase diagnosis of medical conditions by the identification of specific DNA.
  • Target PNA molecules are often present in cell lysates or other source materials in extremely small quantities and in order to amplify such DNA selectively, the polymer chain reaction (PCR) method has been developed.
  • PCR polymer chain reaction
  • a pair of polymerisation primers specific to known sequences of the target DNA are selected, one hybridising at or near the 5' end of the coding strand and the other at or near the 5• end of the non-coding strand such that in the presence of a polymerase, each primer produces a DNA sequence extending the full length of the target DNA template.
  • the DNA so produced is then subjected to strand separation, typically by melting at a temperature of about 90 ⁇ C, the newly formed single stranded DNA sequences will hybridise to excess primer present in the mixture, usually after reducing the temperature to the range suitable for annealing, whereupon in the presence of the polymerase, further DNA strands are synthesised, this time extending only between the termini of the two primers.
  • the polymerase is preferably capable of surviving the high temperature used in the strand separation step, a suitable thermophilic polymerase, namely Taq, having recently become available.
  • the primer(s) permitting immobilization to a solid support or labelling comprise a DNA sequence specific to the target DNA coupled to a non DNA grouping which has to be attached separately by chemical means.
  • the present invention provides, inter alia, a method whereby primer used in the final PCR amplification stage can comprise a non-DNA moiety joined to a standard or general DNA sequence which can be used in the amplification of any target DNA.
  • primer used in the final PCR amplification stage can comprise a non-DNA moiety joined to a standard or general DNA sequence which can be used in the amplification of any target DNA.
  • This is achieved by constructing at least one of the second stage PCR primers with a 'handle' comprising an DNA sequence not hybridising to the target DNA, this handle being specific to a general DNA sequence attached to an inert support or a label so that the latter can be used in a variety of assays and does not need to be synthesised specially for a particular target DNA sequence.
  • the primer(s) having a handle require synthesis to correspond to any particular target DNA, this can be completed on a standard DNA synthesis system without the need to use additional chemical methods for attaching the inert support label. It is particularly useful to be able to use radioisotopes pre-attached to a standard binding ligand or probe and thus to avoid the need for local chemical manipulation of radioactive materials.
  • DIANA immobilised amplified nucleic acids
  • SUBSTITUTESHEET pair are specific to a sequence or sequences of the target DNA between the hybridisation sites of said first primer pair, one of the primers of said second primer pair being immobilised on a solid support or being provided with means for subsequent attachment to a solid support and the other of said second pair of primers carrying a label or being provided with means for subsequent attachment to a label, said amplification being followed by separation of said solid support carrying said amplified DNA and detection of label attached thereto, one or both of said means for subsequent attachment comprising a distal DNA sequence carried by the said primer which does not hybridise with the target DNA but has a selective affinity for a binding partner attached to either a solid support or a label.
  • thermophilic enzyme such as Taq polymerase to permit the above repeated temperature cycle without having to add further polymerase, e.g.
  • the target DNA may be cDNA synthesised from mRNA in the sample and the method of the invention is thus applicable to diagnosis on the basis of characteristic mRNA.
  • Such preliminary synthesis can be carried out by a preliminary treatment with a reverse transcriptase, conveniently in the same system of buffers and bases to be used in the subsequent PCR steps. Since the PCR procedure requires heating to effect strand separation, the reverse transcriptase will be inactivated in the first PCR cycle.
  • ⁇ RNA is the target nucleic acid
  • the oligonucleotide can then serve as a primer for cDNA synthesis, as described in International Patent
  • European 7 Patent Application 192168 of Molecular Diagnostics describes the use of dual probes for hybridisation to target DNA to provide added selectivity, one probe carrying means for immobilisation and the other carrying detection means. However, there is no suggestion of using such probes as primers in PCR amplification.
  • the primer(s) permitting immobilization to a solid support or labelling comprise a DNA sequence specific to the target DNA coupled to a non DNA grouping which has to be attached separately by chemical means.
  • the present invention provides, inter alia, a method whereby primer used in the final PCR amplification', stage can comprise a non-DNA moiety joined to a standard or general DNA sequence which can be used in the amplification of any target DNA.
  • This is achieved by constructing at least one of the second stage PCR primers with a 'handle' comprising an DNA sequence not hybridising to the target DNA, this handle being specific to a general DNA sequence attached to an inert support or a label so that the latter can be used in a variety of assays and does not need to be synthesised specially for a particular target DNA sequence.
  • the primer(s) having a handle require synthesis to correspond to any particular target DNA, this can be completed on a standard DNA synthesis system without the need to use additional chemical methods for attaching the inert support label. It is particularly useful to be able to use radioisotopes pre-attached to a standard binding ligand or probe and thus to avoid the need for local chemical manipulation of radioactive materials.
  • DIANA immobilised amplified nucleic acids
  • a method of diagnosis of medical conditions by identification of specific DNA characterized in that DNA in a sample is subjected to initial amplification by the polymerase chain reaction (PCR) method using a first pair of primers specific to the target DNA and the amplified DNA so produced is further amplified by the PCR method using a second primer pair, one or both of which are different from either of said first primer pair and are specific to a sequence or sequences of the target DNA between the hybridisation sites of said first primer pair, one of the primers of said second primer pair being immobilised on a solid support or being provided with means for subsequent attachment to a solid support and the other of said second pair of primers carrying a label or being provided with means for subsequent attachment to a label, said amplification being followed by separation of said solid support carrying said amplified DNA and detection of label attached thereto, one or both of said means for subsequent attachment comprising a distal DNA sequence carried
  • PCR polymerase chain reaction
  • thermophilic enzyme such as Taq polymerase to permit the above repeated temperature cycle without having to add further polymerase, e.g. Klenow fragment, in each cycle.
  • the target DNA may be cDNA synthesised from mRNA in the sample and the method of the invention is thus applicable to diagnosis on the basis of characteristic mRNA.
  • Such preliminary synthesis can be carried out by a preliminary treatment with a reverse transcriptase, conveniently in the same system of buffers and basics to be used in the subsequent PCR steps. Since the PCR procedure requires heating to effect strand separation, the reverse transcriptase will be inactivated in the first PCR cycle.
  • mRNA is the target nucleic acid
  • the oligonucleotide can then serve as a primer for cDNA synthesis, as described in International Patent Application PCT/89EP/00304.
  • the PCR procedure will be continued, after initial two-stage amplification as described above, by replacing or supplementing at least one primer having such a distal DNA sequence or 'handle* by a third stage primer which hybridises to the handle so that on further amplification the third stage primer will be incorporated into the amplified target DNA.
  • a third stage primer may advantageously be attached to a solid support or may carry means for attachment to a solid support or may carry a label or means for attachment of a label.
  • this third stage primer may advantageously be a standard DNA sequence which will remain the same in all assays irrespective of the target DNA.
  • Both second stage primers may carry 'handles' and in the said third stage of PCR amplification one such primer may be replaced by a primer hybridising to its handle section and being attached to a solid support (or means for attachment thereto) while the other is replaced by a different primer hybridising to its handle and being attached to a label (or means for attachment to a label) .
  • the label may be a radioactive atom such as 32P, C or H which may be incorporated into that one of said second pair of primers.
  • the label may instead be a conventional enzyme or fluorescent substance attached directly to the primer or attached indirectly by, for example, an antibody which binds to the primer.
  • the means for attachment of the solid support or label may be affinity binding group such as biotin/avidin or streptavidin or an amino group or a terminal nucleoside which may interact with a carboxyl labelling of the other end enables the signal from the amplified DNA to be read after each cycle of PCR, or at intervals, thus providing data as to the extent of amplification at each stage. While the signal may be too low to be identified after the first one or two cycles, a plot of signal against PCR cycles can be extrapolated back to origin to give the initial amount of target DNA. Such a procedure requires removal of the immobilised DNA from the medium, while the signal is read, followed by subsequent reintroduction.
  • the immobilising primer is preferably attached to the support initially so that the whole PCR procedure is effected on the support.
  • Magnetic beads provide a particular convenient form of support for this purpose.
  • the label is advantageously a radionuclide to permit rapid reading of the amount of immobilised label.
  • the solid support may, for example, take the form of microtitre wells or dipsticks (e.g. plastic strips) which may be made of polystyrene activated to bind to DNA (K. Aimer, Doctoral Thesis, Royal Institute of Technology, Sweden, 1988). Particles, fibres and capillaries made, for example, of agarose, cellulose, alginate, Teflon or polystyrene are especially convenient.
  • the PCR procedure according to the invention will normally be carried out in a conventional PCR buffer system.
  • the dilution factor is advantageously large, for example in the range 20:1 to 200, e.g. about 100:1; however dilutions, e.g. as low as 1:1 are effective but may
  • SUBSTITUTE SHEET provide less selectivity.
  • the PCR buffer will generally be in the pH range 6.8 to 7.2, Tris/HCL is one suitable buffer. Strand separation is preferably effected at a temperature in the range 90-95°C, annealing of primers in the range 45 to 55 ° C and DNA synthesis in the range 65 to 75"C.
  • the solid support comprises magnetic particles.
  • Preferred magnetic particles are superpara agnetic beads produced by Dynal AS (Oslo, Norway) .
  • One of the second primer pairs may be immobilised on the magnetic particles; the particles may be already attached to the primer during the second PCR amplification stage or may be added subsequently to react with the biotinylated amplified target DNA.
  • the magnetic particles can be added to a mixture containing the target nucleic acid, e.g. a cell extract, stirred and then magnetically drawn to one side of the receptacle.
  • the liquid can then be removed together with unwanted components and the magnetic particles, having the RNA bound thereto, can then be redispersed in a washing solution.
  • the washing step can be repeated several times in quick succession.
  • the whole process of obtaining the target nucleic acid can be performed in under 15 minutes.
  • a further advantage is the ease with which hybridisation or any process effected using the magnetic particles can be continuously monitored by magnetically aggregating the particles at intervals and assaying a label associated either with the material on the particles or with material in the supernatant.
  • the preferred particles are monodisperse and
  • SUBSTITUTE SHEET for example, if two stages of PCR provide double stranded DNA comprising the target DNA terminated with means for strongly bonding to an insoluble support at the 5'-end and a DNA handle for binding to a label via a protein such as Lad at the 3'-end, the amplified target DNA can readily be detected by means of the label, for example by an enzymatic colour reaction, and can then be subjected to strand separation to leave single stranded DNA immobilised on the inset support. The DNA thus immobilised can then be subjected to further operations.
  • synthesis of a labelled second strand can be effected using a labelled primer at the 3'-end, whereupon strand separation liberates labelled ss-DNA into solution.
  • in vitro mutagenesis can be effected using an appropriate mutagenesis primer.
  • the immobilised single stranded DNA may be subjected to sequencing for example as described in International Patent Application WO89/09282 (the contents of which are hereby incorporated herein by reference) e.g. using the method of Sanger et al (Prox. Natl. Acad.Sci. USA 1977, 74., 5462-67). Furthermore, it is possible to omit the initial detection step and carry out sequencing on the immobilised single stranded DNA finally produced by the PCR amplification stages.
  • PCR amplification enables very small amounts of target DNA to be identified and abnormalities thus diagnosed, it is difficult to obtain quantitative information concerning the amount of target DNA present in a sample, due to uncertainty as to the true extent of amplification.
  • Immobilisation of one end of the DNA and labelling of the other end enables the signal from the amplified DNA to be read after each cycle of PCR, or at intervals, thus providing data as to the extent of amplification at each stage. While the signal may be too low to be identified after the first one or two cycles, a plot of signal against PCR cycles can be extrapolated back to origin to give the initial amount of target DNA.
  • Such a procedure requires removal of the immobilised DNA from the medium, while the signal is read, followed by subsequent reintroduction.
  • the immobilising primer is preferably attached to the support initially so that the whole PCR procedure is effected on the support.
  • Magnetic beads provide a particular convenient form of support for this purpose.
  • the label is advantageously a radionuclide to permit rapid reading of the amount of immobilised label.
  • the solid support may, for example, take the form of microtitre wells or dipsticks (e.g. plastic strips) which may be made of polystyrene activated to bind to DNA (K. Aimer, Doctoral Thesis, Royal Institute of Technology, Sweden, 1988) . Particles, fibres and capillaries made, for example, of agarose, cellulose, alginate. Teflon or polystyrene are especially convenient.
  • the PCR procedure according to the invention will normally be carried out in a conventional PCR buffer system.
  • the dilution factor is advantageously large, for example in the range 20:1 to 200, e.g. about 100:1; however dilutions, e.g. as low as 1:1 are effective but may provide less selectivity.
  • the PCR buffer will generally be in the pH range 6.8 to 7.2, Tris/HCL is one suitable buffer. Strand separation is preferably effected at a temperature in the range 90-95*C, annealing of primers in the range 45 to 55*C and DNA synthesis in the range 65 to 75"C.
  • the solid support comprises magnetic particles.
  • Preferred magnetic particles are superparamagnetic beads produced by Dynal AS (Oslo, Norway) .
  • One of the second primer pairs may be immobilised on the magnetic particles; the particles may be already attached to the primer during the second PCR amplification stage or may be added subsequently to react with the biotinylated amplified target DNA.
  • the magnetic particles can be added to a mixture containing the target nucleic acid, e.g. a cell extract, stirred and then magnetically drawn to one side of the receptacle.
  • the liquid can then be removed together with unwanted components and the magnetic particles, having the RNA bound thereto, can then be redispersed in a washing solution.
  • the washing step can be repeated several times in quick succession. The whole process of obtaining Ithe target nucleic acid can be performed in under 15 minutes.
  • a further advantage is the ease with which hybridisation or any process effected using the magnetic particles can be continuously monitored by magnetically aggregating the particles at intervals and assaying a label associated either with the material on the particles or with material in the supernatant.
  • the preferred particles are monodisperse and superparamagnetic and both these properties greatly assist the kinetics of reactions in which the particles are involved. It is a surprising feature of the invention that the probes carried by the particles react in the various reactions virtually as rapidly as if free in solution. Thus, for example, the total isolation of mRNA from a cell lysate using magnetic beads can be effected in about 15 minutes in contrast with the 2 hour period using an affinity column.
  • monodisperse particles that is particles of approximately the same size, the reaction rate and other parameters are particularly uniform.
  • superparamagnetic particles that is particles containing sub-particles of ferromagnetic material which are smaller than the domain size required to maintain permanent magnetism
  • the particles can readily be aggregated at a uniform speed onto a surface by application of a magnetic field but can readily be re-dispersed for a subsequent treatment step, e.g. by physical agitation.
  • This uniformity of behaviour and rapidity of reaction lends itself particularly to automation, which is an essential requirement of many of the nucleic acid manipulations required in commercial production and/or repetitive processes.
  • the preferred magnetic particles for use in this invention are monodisperse superparamagnetic beads produced according to EP 83901406.5 (Sintef) , the disclosure of which is incorporated herein by reference.
  • the iron is very uniformly distributed and provides a very uniform response to a magnetic field which is important in designing a reproducible procedure, particularly for automation, since all the beads move at the same speed.
  • a reproducible amount of iron can be incorporated in each particle, this can be adjusted to a relatively low level which permits the specific gravity of the particles to be in the range specified below.
  • beads having a specific gravity in the range 1.1 to 1.8 most particularly 1.2 to 1.5.
  • the specific gravity is, again, particularly uniform, leading to uniform and predictable kinetic characteristics.
  • the monodisperse particles are spherical beads of diameter at least 1 and preferably at least 2 microns, being preferably not more than 10 and more preferably not more than 6 microns in diameter e.g. about 3 microns.
  • Smaller particles sediment more slowly and in some cases the sedimentation time may be long compared to the reaction time, thus avoiding the need for physical agitation.
  • particles of mean diameter 0.1 to 1.5 microns including fine particles of much smaller diameter behave less reliably in response to magnetisation.
  • the attachment of primers to the particles may be by direct chemical bonding as well as affinity binding, by streptavidin/biotin complexes and the like.
  • the magnetic particles may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups. These may in general be provided by treating uncoated monodisperse, superparamagnetic beads, to provide a surface coating of a polymer carrying one of such functional groups, e.g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an aminoalkylated polymer to provide amino groups.
  • a polymer carrying one of such functional groups e.g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an aminoalkylated polymer to provide amino groups.
  • US Patent No. 4654267 describes the introduction of many such surface coatings.
  • Preferred coated particles for use in the present invention may be prepared by modification of the beads according to the US Patents 4336173, 4459378 and 4654267, the disclosure of which is incorporated herein by reference.
  • macroreticular porous polymer particles prepared from styrene-divinylbenzene and with a diameter of 3.15 microns were treated with HNO- to introduce -NO. groups at the surface of the pores. Then the particles were dispersed in an aqueous solution of Fe 2+. The Fe2+ is oxidised by the -N0 2 groups which leads to precipitation of insoluble iron oxy-hydroxy compounds inside the pores. After heating the iron exists as finely divided grains of magnetic iron oxides throughout the volume of the porous particles.
  • the NO_ groups are reduced by the reaction with Fe to NH_ groups.
  • different monomers are caused to polymerize in the pores and at the surface.
  • the surface carries -OH groups connected to the polymeric backbone through -(CH 2 CH 2 0) 8 ,_ linkages.
  • NH ? groups initially present in the beads may be reacted with a diepoxide as described in US Patent No. 4654267 followed by reaction with methacrylic acid to provide a terminal vinyl grouping.
  • Solution copolymerization with methacrylic acid yields a polymeric coating carrying terminal carboxyl groups as in R452 beads referred to below.
  • amino groups can be introduced by reacting a diamine with the above product of the reaction with a diepoxide as in the R240, R442 and R469 beads, while reaction with a hydroxylamine such as aminoglycerol introduces hydroxy groups as in the M450 and L255 beads.
  • Dynabeads M450 (diameter 4.5 microns) which may be obtained from Dynal, Oslo, Norway have been coated with a monomeric epoxide, resulting in a mixture of epoxy and hydroxy groups. Contact with water however, converts the epoxy groups to hydroxy groups.
  • Dynabeads M-280 are polystyrene beads having hydroxyl groups which have been converted into tosyloxy groups by reaction with p_- toluene sulphonyl chloride.
  • the primer may be attached to the magnetic particles via carboxyl groups, the DNA being firstly provided with a 5'-terminal amino group which can be made to form an amide bond with the carboxyl using a carbodiimide coupling agent.
  • 5'- attachment of DNA can also be effected using hydroxylated magnetic particles activated with CNBr to react with 5'- amino DNA.
  • the 3•-attachment of primer DNA can also be effected by chemical synthesis.
  • the very uniform nature of the monodisperse particles provides uniform reaction rates particularly suited to synthesis in an automated synthesiser such as the Gene Assembler (Pharmacia AS) .
  • the magnetic particle needs to be provided initially with a hydroxyl or protected hydroxyl group.
  • Dynabeads M-280 of Dynal A/S are well suited to this purpose. If necessary, however, other surface functions such as carboxyl could be used to attach a linker carrying a hydroxyl group or alternatively a 3'- attached nucleotide.
  • 5'-Attachment may be effected by coupling of 5'- amino-oligonucleotides to tosyl-activated magnetic particles.
  • the latter may be produced by tosylation of hydroxylated magnetic particles such as Dynabeads M-280 of Dynal A/S. Displacement of the tosyloxy group leaves the 5'-amino group directly attached to the magnetic beads.
  • the 3' end of the probe may be attached to the magnetic particles and this may be conveniently effected by forming a phosphoramidate linkage between the 3'-phosphate grouping of the DNA and an amino group on the particle.
  • biotin labelled nucleotides are commercially available, the 3'- end of DNA fragments can easily be labelled using DNA polymerase and these may be conveniently bound to avidin or streptavidin attached to the magnetic particles e.g. via a hydroxy group.
  • the biotin label may be attached to the nucleotide by a spacer arm, such as one or more e-aminocaproic acid moieties, to minimize steric hindrance.
  • a double stranded plasmid may be cut at a restriction site and end filled with biotinylated nucleotides, thus providing biotin at the 3'-end of each strand.
  • each magnetic particle carries 10 3 -10 6 probes. (1-100 pmols per mg) .
  • the uniform size of the magnetic particles is of advantage in ensuring uniform probe density when the probes are reacted with the particles. Uniform probe density is important in ensuring that all the probes behave in substantially the same way in the various procedures in which they are used.
  • kits for carrying out the method of the invention which will normally include at least the following components:
  • a solid support such as a microtitre well or an array of such wells, a dipstick or beads, more preferably magnetic beads, the support carrying (i) means for attachment to amplified target DNA, (ii) means for attachment to a primer or (iii) a primer;
  • a label carrying means for attachment to amplified target DNA, (ii) means for attachment to a primer or (iii) a primer;
  • a polymerase which is preferably heat stable, for example Taql polymerase;
  • the kit will advantageously contain a substrate for the enzyme and other components of a detection system.
  • the support is a dipstick
  • this may be provided with zones capable of interacting with several different target DNA molecules to enable simultaneous diagnosis of several abnormalities to be performed.
  • zones may carry primers specific to the respective target DNA.
  • Figure 1 shows the oligonucleotides RIT1 to R7 as used in Example 1.
  • Figure 2 shows the extra oligonucleotides RIT8 to RIT11 as used in Example 2.
  • Figure 3 shows diagrammatically the incorporation of biotin and a label into a sequence of target DNA as in Example 1(a).
  • Figure 4 shows the results for Example 1(a) as a plot of relative label activity against PCR cycles.
  • Figure 5 shows diagrammatically the incorporation of biotin and a label as in Example 1(b).
  • Figure 6 shows the results of Example 1(b) as a plot of relative label activity against PCR cycles.
  • Figure 7 shows diagrammatically the incorporation of biotin and a label as in Example 1(c).
  • Figure 8 shows the results of Example 1(c) as a plot of relative label activity against PCR cycles.
  • Figure 9 shows the results of Example 2 as a plot of relative label activity against PCR cycles.
  • Figure 10 shows schematically the use of a DNA-binding fusion protein in the purification and in the detection of DNA fragments.
  • Figure 11 shows schematically the mutagenesis of the lad gene.
  • Figure 12 shows an SDS-PAGE gel of samples from the immobilization and elution of a DNA fragment containing the lac operator sequence as described in Example 3(b) .
  • Figure 13 shows some of the oligonucleotides for S.aureus used in Example 3(c) .
  • the handle primer RIT12 contains the lac operator sequence (as does the RIT11 primer in Figure 2) and RIT6 has a biotin 5'-end.
  • Figure 14 shows the results of Example 3(c) in the detection of S. aureus using PCR amplified DNA and oligonucleotides specific for the staphylococcal protein A gene.
  • Figure 15 shows the results of Example 3(c) in the detection of Streptocoocus G148 using PCR amplified DNA and oligonucleotides specific for the streptococcal protein G gene.
  • Figure 16 shows the oligonucleotide primers used in Example 4 for the detection of the Pfl55 gene of P. falciparum with the annealing site as described by Favaloro et al. (18)
  • Figure 17 shows the results of Example 4(c) in the detection of P. falciparum. Number of parasites per sample as determined by microscopy is shown. The activity is determined as the colour change at 450 nm per minute.
  • Figure 18 shows a schematic drawing outlining the DIANA concept for colorimetric detection of immobilized amplified nucleic acids followed by direct solid phase sequencing.
  • a lacI-lacZ fusion protein is used for the detection and magnetic beads as solid support.
  • Figure 19 shows the sequence of the CrP target gene of C. trachomatis and the primers used for detection (RIT23-26) and sequencing (RIT43) . Mutations determined by genomic sequencing of the clinical samples (Fig.22) are indicated. The numbers refer to the nucleotides as described by Clark et al (20) . Note that the sequence of primers RIT24 and RIT26 are complimentary to the sequence shown. RIT43 hybridises with the lac operator sequence (-TTAACACTCGCCTATTGTTAA-5 • ) which is introduced in the second amplification.
  • Figure 20 shows an agarose gel demonstrating the binding of the amplified fragment to the beads and the size of the inner and outer fragment.
  • Lanes 1 and 2 are from material from the second amplification and lanes 3 and 4 are from the outer fragment. Lanes 1 and 3 are from unbound material while lanes 2 and 4 correspond to the supernatant after the binding to the beads.
  • the marker is ⁇ DNA digested with Pst I. Note that the length of the fragments is the expected size (267 and 421 base pair respectively) .
  • Figure 21 shows the results of the DIANA assay on clinical chlamydia samples.
  • the results of the clinical assay performed by a standard cell culture technique (35) is indicated (+/-) .
  • the activity of the DIANA is defined as described in Materials and Methods.
  • Sample 8 is a positive control of cultivated C. trachomatis and sample 9 is a negative control for the PCR reactions (no DNA template was added to this tube) .
  • Figure 22 shows the results of DNA sequencing of one (number 1, Fig. 21) of the positive chlamydia samples. The sequence was performed as described in Figure 18 with a general RIT43 fluorescent labelled primer. The analysis was performed on an ALF automated laser fluorescent sequencer (Pharmacia, Sweden) as described by the manufacturer. MATERIAL AND METHODS
  • Bacterial strains and plasmids Escherichia coli HB101 (1) and JM103 (2) were used as bacterial hosts.
  • the plasmid vectors used were pSI.l (3), pEMBL9 (4), pDMI.l (5), pNSEQl (6), pEZZT308 (7) and pSKS104 (8).
  • M13K07 (9) was used as helper phage during mutagenesis.
  • Staphylococcus aureus SA113 (10) , Streptococcus G148 (11) and Bacillus subtilis 168 (12) were used in the Examples. These were grown as single colonies on TBAB-plates (Difco, USA) at 37*C overnight.
  • a strain of C. trachomatis biovar L2 was kindly supplied by H. Gnarpe (Gavle Hospital, Sweden) . Clinical samples were obtained with cotton-tipped swabs from male urethral (Karolinska Hospital, Sweden) and stored in PCR buffer (see below "PCR amplification") at +4"C. Plasmodium falciparum parasites from patient blood samples were prepared as described by Zolg et al., (13), and the microscopic analyses were preformed according to the giemsa stained blood smears method (14) . The strains and plasmids have been deposited at the Department of Biochemistry, Royal Institute of Technology, Sweden. Synthesis of oligonucleotides
  • oligonucleotide primers complimentary to the staphylococcal protein A gene or the streptococcal protein G gene (see Figure 1, 2 and 13) , were synthesized by phosphoramidite chemistry on an automated DNA synthesis machine (Gene Assembler, Pharmacia, Sweden) as described by the manufacturer. Two of these primers (RITl and RIT6) was synthesized with an amino group in the 5 -end, which was subsequently used to introduce a biotin-derivative (15) as described by the manufacturer (Pharmacia, Sweden). RIT2, RIT7, RIT11
  • oligonucleotide primers complementary to Chlamydia trachomatis (20)
  • four primers complementary to P. falciparum (21) (RIT33-36)
  • a general sequencing primer (RIT43) were synthesized as described above.
  • Two primers (RIT25 and RIT43) were synthesized with an amino group in the 5'-end, which was used to introduce a biotin (Clonetech, USA) and a fluorescent group (Pharmacia, Sweden) , respectively, as described by the manufacturer.
  • RIT25 and RIT43 were purified using a FPLC pepRPC 5/5 column (Pharmacia, Sweden) .
  • Cell extracts were obtained by sonication (18) ; the fusion protein, lacI-SPA, was purified by IgG affinity chromatography using IgG Fast Flow Sepharose (Pharmacia) as recommended and eluted, with 0.3 M HAc, pH 3.3. Fractions were collected and lyophilized prior to SDS-PAGE analysis with a 3.5% stacking gel and a 13% separating gel; performed according to Laemelli (19). The gels were stained with Coomassie blue.
  • Recombinants containing a functional lacZ gene were assayed by plating on X-Gal (5-bromo-4-chloro- 3-indolyl- ⁇ -D-galactoside) plates as described earlier (16) .
  • ⁇ -Galactosidase was assayed by as colorimetric procedure using ONPG (o-nitrophenyl- ⁇ -D-galactoside) in accordance with the supplier's recommendation.
  • Alkaline phosphatase was assayed at 37"C with 15.2 mM p-nitrophenyl phosphate (Sigma No. 104-0) as substrate in a buffer consisting of 0.1 M glycine and 1 mM MgCl p , pH 10.4.
  • the streptavidin-alkaline phosphatase conjugate was supplied by Boehringer Mannheim.
  • the in vitro amplification was carried out on a Techne Programmable Dri-block PHC-l (Techne, UK) .
  • the general PCR solution was 1 mM of each of the primers, 200 uM of each dATP, dCTP, dGTP and dTTP, in a solution containing 67mM Tris-HCl, pH8.8, 16.6 mM (NH ) SO , 6.7 mM MgCl 2 , lOmM ⁇ -mercapto- ethanol and 170 ug/ l BSA under a layer, of paraffin oil.
  • a single colony was picked from the TBAB-plate with a sterile pasteur-pipette and put into a 0.5 ml microfuge tube (Kemila, Sweden) containing 10 ul of the PCR solution adjusted to pH 10 with NaOH. The tube was transferred to the temperature block and the cells were lysed with a 5 minute heating at 95 'C followed by cooling to room temperature.
  • the solution was thereafter adjusted to pH 8.8 by adding an equal amount of a solution containing 67 mM Tris-HCl, pH7, 16.6 mM (NH 4 ) 2 S0 4 6.7 mM MgCl , 10 mM ⁇ -mercaptoethanol, 170 ug/ml BSA and 1 unit of Taql DNA-polymerase (Stratagene, USA) .
  • a temperature cycle was started consisting of the following program: denaturation of template at 92'C for 1 minute; annealing of primers at 50 * C for 2 minutes and extension of primers at 72*C for 1 minute. After each fifth repeated cycle, 5 ul of solution was transferred to a new tube for binding to magnetic beads. Binding to magnetic beads
  • Magnetic beads containing covalently coupled streptavidin were obtained from Dynal (Oslo, Norway) . After the in vitro amplification using PCR, 5 ul of solution was added to a solution containing 10 ul 10 mM Tris-HCl, pH 7.5 and l mM EDTA with 158 ug of magnetic beads. The solution was incubated for 30 minutes at room temperature and non-bound DNA was removed by repeated washes with 35 ul of 5 M NaCl followed by 10 mM Tris-HCl, ImM EDTA, pH 7.5. A neodymium-iron-boron permanent magnet (Dynal, Oslo) was used to hold the beads in the tube during this procedure. Before the final wash, the beads were transferred to a new tube to avoid background binding of radioactive label. Detection of radioactive label
  • RITl is biotinylated at the 5'-end, while RIT2 has been labelled at the 5'-end with 32P.
  • Three sets of experiments using different bacterial cells were carried out using these primers specific for the staphylococcal protein A gene; first S. aureus cells containing the protein A gene and then two control cells, B.subtilis and Streptococcus. lacking this gene.
  • the results for 35 cycles of PCR followed by immobilization to magnetic beads are shown in Figure 4. As shown an increase of incorporated label is obtained during the PCR reaction. However, a high background is also obtained for the control samples (B.subtils and Stretococcus G148) .
  • Amplification of the specific DNA sequence was carried out according to the invention.
  • the concept, designated dual polymerase chain reaction (D-PCR) is shown schematically in Figure 5.
  • a pair of primers specific for a sequence of the cell to be detected is used for a first round of in vitro amplification. After a suitable number of temperature cycles (e.g. 5 to 30 cycles), the sample is diluted e.g. 1:1 and a new primer pair is added. These primers are complimentary to internal parts of the primary amplified DNA fragment.
  • the second pair has at the 5'-end either an affinity handle (such as biotin) or a label (such as an isotope) . After additional PCR cycles, the amplified material is bound to a solid support and the label detected.
  • the rationale behind the concept is that a high amount of specific template for the second PCR reaction is obtained during the first PCR reaction. This reduces the non-specific amplification of biotin and label containing DNA fragments as seen in example 1(a).
  • the oligonucleotides RIT3 and RIT4 were used for the first PCR reaction. However, lower concentration of primers were used (0.2 mM of each primer) and only 25 temperature cycles were performed. After 25 cycles, the mixture was diluted 1:1 with the buffer solution containing nucleotides and additional 1 unit of Taq polymerase was added together with 1 mM of the oligonucleotides RITl and RIT2, having biotin and 32P at their respective
  • This example illustrates that a high degree of labelling can be introduced into the PCR amplified material and that it can be detected using solid- phase technology described here. Low background label is achieved by the use of nested primers.
  • RIT3 and RIT4 were used for a first round of PCR as described in (b) . After 25 cycles, the mix was diluted 1:1 with the buffer solution containing nucleotides and additional 1 unit of Taq polymerase was added together with 1 mM of the oligonucleotides RITl, RIT2, RIT6, with biotin in the 5-end and RIT2 with 32 P in the 5-end. After various cycle times, 5 ul of PCR mix was taken out and allowed to bind to magnetic beads containing streptavidin as described above.
  • Streptococcus G148 is obtained, while a high degree of label is obtained for the specific cells (S.aureus) after 15 cycles.
  • the detection of the S.aureus gene was carried out as described in Example 1 except that the second round of PCR contained only three oligonucleotides; RIT5, RIT6 with biotin and RIT2 end labelled with 32 P.
  • the detection of the Streptococcus gene was carried using the same scheme as described above using primers RIT8 and RIT9 for the first round of PCR (25 cycles) and RIT10, RIT6 with biotin and RIT11 end labelled with 32 P for the second round of PCR.
  • the second round of PCR was terminated after 15 cycles and the amplified material was allowed to bind to streptavidin coated magnetic beads as described in Example 1.
  • This Example concerns the use of a DNA-bindihg protein to purify DNA fragments and in the detection of PCR amplified fragments as shown schematically in Figure 10.
  • E.coli JM103 After transformation, E.coli JM103, was spread on plates consisting of ampillicin, X-Gal and IPTG, and white colonies were isolated.
  • Single strand plasmid DNA, pEMBL/lacI was obtained by transforming plasmid DNA from one of these colonies into JM103 using the packing deficient helper phage M13K07 to infect and to pack single stranded DNA with the pEMBL-vector carrying the organ of replication from phage fl.
  • a synthetic oligonucleotide (5'-ATTCCCGGGATCCTCTGCCOGCTTTCCAG-3') a mismatch priming on the single strand template was performed.
  • the primer extension conditions were essentially as described by Carter (17) .
  • the extended material was used to transform E.coli JM103.
  • the mutant plasmids, pEMBL/lacI STOP, were selected by blue colonies on agar plates containing ampicillin, X-Gal and IPTG.
  • Figure 11 shows the in vitro mutagenesis schematically; the nucleotides indicated by asterisks are those which are deleted during the mutagenesis.
  • the 3•-end of the mutated lacl gene without the stop codon was released with EcoRV and Xma from plasmid pEMBL/lacI STOP yielding a fragment of 291 bp.
  • the isolated fragment was ligated into pDMI.l, which had been digested with the same restriction enzymes.
  • the pDMI.l plasmid consists of three major elements: a lacl ⁇ gene overproducing the lac repressor, the gene for neomycin phosphotransferase conferring resistance to kanamycin and the pl5A replicon.
  • the resulting construction, pDMI.l STOP thus encodes mutant lad.
  • a fragment encoding a part of protein A was cleaved out from the pNSEQl vector with BamHI and isolated. This fragment was then inserted into a unique BamHI site in the 3'-end of the lacl-gene in plasmid pDMI.l STOP plasmid. This new plasmid, pRIT34, encodes the lacI-SPA fusion gene of 1926 bp.
  • E.coli strain HB101 harbouring the plasmid pRIT34 was grown over night in baffled Erlenmeyer- flasks containing Tryptic Soy Broth (30 g/1) (Difco, USA) with an addition of Yeast Extract (7 g/1) , and kanamycin (50 mg/1) .
  • the fusion protein, with a molecular weight of 70.6 kDA (deduced from the amino acid sequence) was purified from sonicated cells using affinity chromatography on matrix-bound IgG. After elution and lyophilizing the affinity-purified proteins were analyzed by SDS-PAGE. The production level was approximately 20 mg/1 culture. Almost 90% of the affinity purified protein was found to be full-length.
  • This example illustrates that a lacI-SPA fusion protein can be produced in a re ⁇ ombinant host and that the fusion protein can be immobilized and purified on a solid support containing covalently bound IgG.
  • (b) Purification of DNA fragments Plasmid DNA of pEZZT308 was digested with HgiAI and Notl, yielding fragments of different length: 85, 497, 676, 1161 and 1183 bp. The fragment of 676 bp contained the lac operator sequence. After dilution of the digested DNA to a concentration of 140 ng/ul it was divided into 3 aliquotes.
  • IgG-Sepharose was mixed with lysates of HB101 ' harbouring pRIT34 for 1 hour at room temperature.
  • the lacI-SPA fusion protein present in the lysate became immobilized on the IgG-Sepharose via the SPA moiety.
  • 100 ul of the immobilized lac-SPA fusion protein was mixed at room temperature with 100 ul of digested DNA from plasmid pEZZT308. Analysis of the supernatant revealed that the 676 bp fragment had selectively bound to the gel ( Figure 12) , lane 3) .
  • the streptavidin- alkaline phosphatase is bound to the amplified DNA via the 5' biotin, as shown in Figure 10B. Excess of conjugate was eliminated by three washings with 1.5 ml of washing buffer. 500 ul of alkaline buffer was then added, the temperature adjusted to 37"C, and then 500 ul of substrate was added. The enzyme reaction was stopped with 40 ul 0.5 M NaOH and the activity measured (by change of absorbence per second at 405 nm) using a spectrophotometer.
  • Figure 15 and 16 show the results of using either oligonucleotides specific for S.aureus ( Figure 14) or oligonucleotides specific for Streptococcus G148 ( Figure 15) .
  • a high degree of colour change above a background level is achieved for both oligonucleotides.
  • the DIANA was used to determine the presence of Plasmodium falciparum DNA in clinical blood samples.
  • the two step PCR procedure outlined in Figure 10 was carried out using the five oligonucleotides shown in Figure 16.
  • the P. falciparum specific primers hybridize to the 5'-end of- exon II in the Pfl55/RESA gene (21).
  • This gene codes for a parasite antigen, considered to be one candidate for a future malaria vaccine (22,23).
  • the Pfl55/RESA gene has only been characterized in two parasite strains earlier (21). The 5' region of the gene was chosen because it is thought to be relatively conserved in different strains, although it might contain minor variations.
  • the first set of primers generates a 428 bp fragment and the second set a 398 bp fragment in the earlier characterized strains.
  • the two step PCR amplification was carried out on 14 samples obtained from patients in The Gambia, Africa as described in Example 3.
  • the resulting material was captured in the Lacl containing sepharose (Example 3) and detected via the streptavidin-alkaline phosphatase conjugate.
  • the results of the assay are presented in Figure 17. Note that samples from patients with low grade parasitemias were included in the study, to investigate the sensitivity of the assay. All the samples positive by microscopy, were also positive in the DIANA, using only 1 ⁇ l sample volumes. 10 parasite genomes could reproducibly detected, with a distinct signal significantly over background. EXAMPLE 5
  • This example describes a system for rapid colorimetric detection of specific genomic DNA fragments amplified by the polymerase chain reaction (PCR) which has been designed to allow for direct solid phase sequencing of positive samples.
  • the amplified material is immobilized to magnetic beads using the biotin streptavidin system.
  • a lac operator sequence is incorporated in the amplified material during the second step of a nested primer procedure.
  • This 21 base pair sequence is used for a general colorimetric detection with a fusion protein consisting of the Escherichia coli lac repressor and 0-galactosidase. Positive samples can subsequently be treated with alkali to obtain a single stranded DNA template suitable for direct genomic sequencing.
  • DIANA immobilized amplified nucleic acids
  • the material obtained after the first PCR step is diluted and subsequently used for a second PCR with the inner primers, which are specific for a sequence within the DNA fragment amplified in the first step.
  • One of the primers is biotinylated, while the other contains a lac operator "handle" consisting of 21 nucleotides.
  • successful amplification yields specific DNA with biotin and lac operator incorporated in the fragment.
  • the second PCR reaction is carried out with fewer cycles, 15, and therefore very low yield of fragments containing biotin and lac operator will be obtained for samples containing non-specific DNA (Fig. 18) .
  • the biotinylated material is thereafter captured on magnetic beads coupled with streptavidin using interaction between biotin and streptavidin.
  • a recombinant fusion protein consisting of the lac repressor (lad) and / 8-galactosidase (lacZ) is added to the beads and bound enzyme conjugate is detected by adding chromogenic substrate, specific for the ⁇ - galactosidase enzyme.
  • the samples identified as positive using this colorimetric procedure can directly be treated with alkali to remove the bound fusion protein and to melt the double stranded DNA immobilized to the beads.
  • the biotin streptavidin complex is resistant to this treatment and thus a single stranded template suitable for sequencing is obtained using this one-step elution procedure.
  • a solid phase DNA sequencing protocol (24) can therefore be followed without the need for extra template preparations.
  • the extended material is finally eluted from the beads using formamide and loaded on a sequencing gel.
  • Chlamydia trachomatis is Gram negative bacteria characterized by an obligatory parasite life cycle within eucaryotic host cells.
  • the human C. trachomatis isolates have been grouped in two biovars and 15 serovars (25,26) Serovars LI, L2 and L3 are clinically important as they are associated with relatively invasive from of chlamydia disease involving lymphoid tissue (27) .
  • oligonucleotide primers specific for the middle part of the CrP coding region of C. trachomatis serovar L2 were synthesized. The sequence of the four primers are shown in Figure 19 where also the location of the target DNA within the CrP gene is shown. The two outer primers hybridize 421 base pairs from each other, while the inner primers are 267 base prais apart. Biotin was covalently coupled to one (RIT25) of the inner primers, while the 21 base pair handle corresponding to the E.coli lac operator sequence (see Material and Method for details) was added to the 5'-end of the other inner primer (RIT26) .
  • a culture of C. trachomatis serovar L2 was used for a standard agarose assay.
  • Cells were directly lysed in the PCR buffer and the CrP gene was amplified for 25 cycles using the outer primers (RIT23 and RIT24, Fig. 19).
  • a second PCR step was performed for 15 cycles using the inner primers (RIT25 and RIT26, Fig. 19).
  • the clinical tops were put into PCR tubes filled with PCR buffer (10 ⁇ l>. and rubbed against the wall of the tube. The tube was subsequently heated to 99*C for 5 minutes in order to lyse the cells and then cooled on ice.
  • the lysed mixture 90 ⁇ l of a PCR buffer with 0.2 mM dNTP, 0.2 mM of each primer and 1.0 units of Taq polymerase (Boehringer Mannheim, Sweden) was added.
  • One temperature cycle on the PCR was: denaturation of template 95 ' C, 0.5 minute; annealing of primers 58*C, 1 minute; extension of primers 72*C, 1 minute.
  • Magnetic beads with covalently coupled streptavidin, Dynabeads M280-Streptavidin (31) was obtained from Dynal (Norway) .
  • a neodymium-iron-boron permanent magnet (Dynal, Norway) was used to sediment the beads during washing procedure.
  • 300 ⁇ g of the beads was mixed with 85 ⁇ l of the PCR mixture and incubated for 20 minutes at room temperature. The beads was then extensively washed using the TST buffer, described above.
  • the results (Fig. 20) shows that the first PCR yields a specific band (lane 3) of the expected size (421 base pairs) , which does not bind to the magnetic beads (lane 4) .
  • the fusion protein was purified by (NH 4 ) 2 S0 4 precipitation, using the fraction between 25 and 45% (NH 2 SO ⁇ , saturation, followed by affinity binding to Heparin- Sepharose column (Pharmacia, Sweden) .
  • the fusion- protein was eluted in a 0.25 M KCl and 15% glycerol buffer.
  • Example 5(c) The 9 samples were amplified as described in Example 5(b) and the obtained material was immobilised to Dynabeads M280-streptavidin.
  • the beads was washed with the TST-buffer.
  • the substrate, ortho- nitrophenyl- / S-D-galactoside (ONPG) was added and the change of absorbence at room temperature was analyzed.
  • One unit is defined as the change of one A unit per minute at 405 nm.
  • a buffer containing: 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 and 0.1 mg/ml BSA was mixed together with 2 pmole RIT43 primer in a total volume of 17 ⁇ l. The annealing mixture was heated at 65°C and allowed to cool to room temperature.

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Abstract

Le procédé et le kit décrits, qui servent à diagnostiquer des conditions médicales par identification d'un ADN spécifique, se caractérisent en ce qu'un ADN contenu dans un échantillon est soumis à une amplification initiale par le procédé de la réaction en chaîne par polymérase (PCR) grâce à l'utilisation d'une première paire d'activateurs spécifiques à l'ADN cible, et l'ADN amplifié ainsi produit est amplifié encore davantage par le procédé PCR grâce à l'utilisation d'une seconde paire d'activateurs, dont l'un et/ou l'autre sont différents de ceux de la première paire d'activateurs et sont spécifiques à une séquence ou à des séquences de l'ADN cible entre les sites d'hybridation de la première paire d'activateurs. L'un des activateurs de la seconde paire d'activateurs est immobilisé sur un support solide ou est pourvu d'organes permettant son attache ultérieure sur un support solide et l'autre activateur de la seconde paire d'activateurs porte une marque ou est pourvu d'organes permettant son attache ultérieure à une marque. L'amplification est suivi par une séparation dudit support solide portant l'ADN amplifié et par une détection de la marque attachée à lui. L'un et/ou l'autre de ces organes servant à l'opération d'attache ultérieure comprend une séquence d'ADN distalle portée par ledit activateur qui ne s'hybridise pas avec l'ADN cible mais présente une affinité sélective pour un partenaire de liaison attaché soit à un support solide soit à une marque.
PCT/EP1990/000454 1989-03-22 1990-03-15 Diagnostic en phase solide de conditions medicales WO1990011369A1 (fr)

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JPH04505999A (ja) 1992-10-22
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JP3152656B2 (ja) 2001-04-03
DE69029726D1 (de) 1997-02-27
AU5272890A (en) 1990-10-22
CA2050681A1 (fr) 1990-09-23
ATE147791T1 (de) 1997-02-15
EP0464067B1 (fr) 1997-01-15
DE69029726T2 (de) 1997-09-04
KR920700360A (ko) 1992-02-19

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